Abstract Respiratory syncytial virus (RSV) is among the leading causes of pediatric death secondary to pneumonia worldwide. Although the molecular biology of RSV has been elucidated in detail, specific mechanisms of pathogenesis remain poorly defined. Historically, the study of RSV, both in cell culture and animal models has relied on a relatively small number of laboratory strains, many of which have been passaged in culture for over 50 years. We propose an innovative approach to the study of RSV pathogenesis that includes the use of clinical isolates. Our hypothesis is that clinical isolates of RSV possess naturally occurring mutations (polymorphisms) that result in alteration in pathogenesis and virulence. Indeed, we have identified clinical isolates of RSV that differ significantly in their ability to induce cytokines and the innate immune response in human macrophages. Specific aim 1 is to map these induction phenotypes on the viral genome. To do this, we have created an infectious clone of RSV strain NH1125B, a high inducing strain, and have recovered recombinant virus. We will use this system to recover recombinant viruses in which regions of the NH1125B genome have been replaced with corresponding regions of the genome of NH1067B, a low inducing strain. Chimeric viruses will be screened for their ability to induce cytokine production and host innate immunity using transcriptome analyses. Specific Aim 2 is to define the innate immune responses in children infected with RSV and to determine whether the patterns of innate immune activation are associated with specific genotypes of RSV. To do this, we will conduct a clinical study and enroll RSV-infected children < 1 year old with either mild disease (evaluated in the clinic or emergency department and discharged home) or severe disease (admitted to the Pediatric Intensive Care Unit (PICU) with severe respiratory tract illness) and determine A) whether the genetic elements identified in Specific Aim 1 are associated with severity of RSV disease. Here our hypothesis is that the viral polymorphisms defined in Specific Aim 1 will be unequally distributed between the viruses obtained from the children in each group. B) We will determine whether the transcriptome profiles of cells present in respiratory secretions of children with either mild or severe infection are distinct and whether these transcriptome patterns are associated with viral genotype. This will be accomplished by defining the transcriptome, by RNA-SEQ technology, of host cells in respiratory secretions of RSV-infected children. We have developed a means to obtain nearly complete virus genome sequence from respiratory specimens using RNA-SEQ. Using bioinformatics, we will determine whether viral genetic genotype are associated with host gene expression. In all, the findings of these studies will shed light on the genetic elements of the RSV genome that are associated with severity of disease and pathogenesis. Ultimately, the data obtained here will provide novel approaches to RSV vaccine and antiviral therapy.